PROJECT SUMMARY Clostridium difficile infection (CDI) is a leading nosocomial infection and the primary identifiable cause of antibiotic-associated diarrhea. It can lead to multiple CDI recurrences, sepsis, toxic megacolon, and death. Unfortunately, current antibiotic approaches do not always prevent these outcomes. This proposal focuses on a new approach to reducing CDI severity through exploiting an endogenous lipid signaling pathway that we recently found protects mice against the damaging effects of CDI. Prostaglandins (PGs) are endogenous lipid mediators generated by the cyclooxygenase (COX)- dependent metabolism of arachidonic acid. PGE2 is abundant at sights of inflammation and infection, and supports colon epithelial cell health primarily through ligation of the E Prostanoid (EP)4 receptor. In contrast, COX inhibitors, known as nonsteroidal anti-inflammatory drugs (NSAIDs), are among the most widely consumed drugs available and are known to cause gastrointestinal side effects. Epidemiological data link NSAID use to various forms of colitis, including CDI. Our new preliminary data show that prior exposure to NSAIDs worsens subsequent CDI in a mouse model, while a commonly-prescribed, stable PGE1 analogue (misoprostol) can alleviate it. The mechanism whereby prostaglandins provide protection against CDI is unclear, but PGE2 has long been studied for its effects on promoting colon epithelial cell survival, in large part by transactivating the epidermal growth factor receptor (EGFR). We have preliminary data to show that PGE2 prevents colon epithelial cell death in the setting of C. difficile intoxication, in an EGFR-dependent manner. These data lead us to the hypothesis that PGE2 protects the gastrointestinal tract during CDI through EP4 dependent transactivation of the EGFR in intestinal epithelial cells, thereby reducing severity of CDI. We plan to test this hypothesis by (1) defining the role of PGE2 in limiting CDI severity and (2) elucidating the PGE2- stimulated signaling pathway in intestinal epithelial cells that is required for protection against CDI. These studies are a critical first step in moving toward clinical studies of CDI treatment and/or prevention that exploit the beneficial effects of PGE2 on colon health. The successful completion of these studies will have immediate impact on new clinical studies of CDI may provide evidence for more rationale use of NSAIDs in high CDI-risk patients.